PROJECT SUMMARY Increased microvascular permeability is central to the pathogenesis of sepsis and multi-organ system dysfunction. Our group has identified cell-free hemoglobin (CFH), hemoglobin that has escaped from the antioxidant confines of the red blood cell (RBC), as a major proximal trigger of increased microvascular permeability in sepsis. The increase in circulating CFH in sepsis is thought to be due to sepsis-induced red cell fragility. Our parent R01 tests the hypothesis that CFH is a key endogenous mediator of sepsis-induced ARDS through its induction of endothelial mitochondrial dysfunction, leading to increased pulmonary microvascular permeability. Our initial studies are well underway to test this hypothesis, focusing on the downstream effects of CFH on the endothelium. However, the determinants of increased RBC fragility and increased endothelial susceptibility to CFH-mediated injury during sepsis are unknown. Published data and our preliminary studies point to ascorbate, Vitamin C, as a key regulator of both RBC fragility and endothelial sensitivity to injury in sepsis. Low RBC ascorbate concentrations increase red blood cell fragility in diabetes, priming the red cells to hemolyze and release CFH, but the effects of ascorbate on RBC fragility in sepsis has never been studied. Our group recently reported that ascorbate depletion of primary human endothelial cells makes them more susceptible to CFH-mediated increases in paracellular permeability and that restoring intracellular ascorbate concentrations attenuates this effect. Thus, low ascorbate concentrations in sepsis have the potential to both increase RBC fragility and prime the endothelium for increased permeability. In additional preliminary studies, we found that supplementation of ascorbate in an isolated perfused human lung model attenuated the increased vascular permeability induced by CFH. With these compelling studies as a foundation and the support of this NHLBI administrative supplement, we will address two critical knowledge gaps: (1) what are the effects of intracellular ascorbate levels on RBC fragility and CFH release and (2) do intracellular ascorbate levels modify the effects of CFH on endothelial permeability in sepsis? These questions are timely as there are several ongoing clinical trials of ascorbate supplementation in sepsis with a paucity of mechanistic, pre-clinical data to support this therapeutic approach. In this application we will fill these knowledge gaps by testing the overarching hypothesis that intracellular ascorbate depletion in sepsis is a key mediator of both increased hemolysis and CFH-mediated increases in microvascular permeability. This hypothesis will be tested within the scope of the parent grant with the following two Aims: (1) To test the hypothesis that low intracellular levels of RBC ascorbate increase RBC fragility in sepsis, and (2) To test the hypothesis that circulating and tissue levels of ascorbate are decreased in a mouse model of sepsis, augmenting the effects of CFH on increased endothelial permeability. This project is within the scope of the parent proposal, will answer key questions in the sepsis field and will address a major goal of the Office of Dietary Supplements.